Edible scaffold for cell culture of cultured meat and Manufacturing method thereof

Abstract

The present invention relates to a support for cell culture of edible cultured meat and a method for manufacturing the same. More specifically, the invention relates to an edible support for cultured meat cells that is edible and has a meat-like texture, a method for manufacturing the same, a method for manufacturing cultured meat using the same, and an invention capable of providing a processed food product thereof.

Description

Technology Field

[0001] The present invention relates to an invention capable of providing a support for cell culture of edible cultured meat, a method for manufacturing the same, a method for manufacturing cultured meat using the same, and a processed food product thereof. Background Technology

[0003] With increasing concern over environmental pollution caused by the livestock industry and the dignity of life, the number of people seeking cultured meat (or alternative meat) is rising worldwide. Cultured meat refers to meat intended to replace natural meat, specifically meat produced by culturing stem cells or similar substances in a culture medium. While many products utilizing textured vegetable protein (TVP) have been released on the market to create existing meat substitutes, there is a problem in that these products fail to replicate the texture of actual meat when tasted.

[0004] Replacing the texture of meat requires a high level of technology and has therefore been considered a difficult challenge to date. One method to mimic meat texture is to implement a grain in meat substitutes; to this end, the U.S. company 'Beyond Meat' has proposed a method of extruding proteins isolated from soybeans. The core technology lies in creating dense isolated proteins by controlling pressure, temperature, and speed during the extrusion process. Consequently, significant investment is being made worldwide in the development of extruders, with research being conducted particularly on technologies capable of precisely controlling high pressure. However, achieving the desired meat texture from plant-based proteins using these methods remains a major challenge.

[0005] In particular, there have been various attempts to achieve a meat-like texture using TVP-type materials.

[0006] Generally, scaffolds made from non-edible materials used in cultured meat production require an essential process of separating the cultured cells after cell proliferation and culture; however, there is a problem in that cell destruction is induced during separation, leading to a deterioration in the quality of the final cultured meat.

[0007] In addition, in the case of scaffolds manufactured using conventional edible raw materials, they are produced by 3D printing using gel materials, but they have a jelly-like texture in terms of texture, which is a limitation as they do not have the texture of meat. In the case of scaffolds using TVP, the strength decreases during the cell culture period, so they cannot maintain the shape of the scaffold, which has a very narrow range of applications. Prior art literature

[0009] Chinese Published Patent No. 113647568 (Publication Date: Nov. 16, 2021) Korean Published Patent No. 10-2023-0034115 (Publication Date: Mar. 09, 2023) Korean Registered Patent No. 10-2151203 (Publication Date: Sep. 02, 2020) The problem to be solved

[0010] The present invention aims to provide an edible support for cell culture of cultured meat that satisfies the physical properties required for a support used in the production of cultured meat, is edible, has a meat texture, and eliminates the need for separate separation of cultured cells from the support, a method for producing the same, and a method for producing cultured meat using the same.

[0011] Accordingly, the technical problems to be solved by the present invention are not limited to those mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0013] The present invention, for solving the above problem, relates to an edible support for cell culture of cultured meat, comprising: an extruded product formed by extruding textured vegetable protein (TVP) raw material and water; and a collagen modified layer on the surface of the extruded product.

[0014] As a preferred embodiment of the present invention, the TVP raw material may be used comprising 20.50 to 22.50 weight% carbohydrates, 62.20 to 66.50 weight% crude protein, 1.50 to 2.00 weight% crude fat, and the remaining amount of moisture and ash.

[0015] As a preferred embodiment of the present invention, the extruded product may have a length of 3 mm or more and a cross-sectional diameter of 9 mm for the long side and 7 mm for the short side before hydration, and a length of 3 mm or more and a cross-sectional diameter of 10 mm for the long side and 8 mm for the short side after hydration. At this time, the hydration is performed for 24 hours under conditions of immersion in water at 20 to 27°C and stirring at 50 rpm using a vertical mixer.

[0016] As a preferred embodiment of the present invention, the edible support has a length of 3 mm and a cross-sectional area of ​​251 mm 2 When, the total average pore area in the edible support is 10,000 µm 2 It could be more than that.

[0017] As a preferred embodiment of the present invention, the total average area of ​​pores within the hydrated edible support measured after hydrating the edible support for 24 hours can be increased to 10.0% or less.

[0018] As a preferred embodiment of the present invention, when measuring the compressive strength by compressing the edible support to 75% of its length or height using a Brookfield CTX measuring instrument, the compressive strength (TA hardness) may be 100 to 300g.

[0019] As a preferred embodiment of the present invention, the compressive strength measured after hydrating the edible support for 21 days may have a compressive strength reduction rate of less than 76.0% according to the following mathematical formula 1.

[0020] [Mathematical Formula 1]

[0021] Compressive strength reduction rate (%) = (Compressive strength of edible support before hydration - Compressive strength of edible support after 21 days of hydration) / (Compressive strength of edible support before hydration) × 100%

[0022] In mathematical formula 1, the above compressive strength was measured by compressing the edible support to 75% of its length or height using Brookfield’s CTX, and the above hydration was performed for 21 days under conditions where the edible support was immersed in water at 20–27°C and stirred at 50 rpm using a vertical mixer.

[0023] As a preferred embodiment of the present invention, the edible scaffold may satisfy the following Equation 1 when measuring the cell adhesion rate of muscle stem cells to the edible scaffold after suspension culture of muscle stem cells at 37°C and 5 volume% CO2.

[0024] [Equation 1]

[0025] 500% ≤ (Cell adhesion rate on day 14 / Cell adhesion rate on day 7) × 100% ≤ 800%

[0026] As a preferred embodiment of the present invention, the edible scaffold may satisfy the following Equation 2 when measuring the cell adhesion rate of muscle stem cells to the edible scaffold after suspension culture of muscle stem cells at 37°C and 5 volume% CO2.

[0027] [Equation 2]

[0028] 600% ≤ (Cell adhesion rate on day 21 / Cell adhesion rate on day 14) × 100% ≤ 1200%

[0029] As a preferred embodiment of the present invention, the collagen modified layer may be formed by surface modification treatment with a surface modifying agent comprising collagen powder having a molecular weight of 10 to 250 kDa and acetic acid at a concentration of 0.05 to 1.0 volume%.

[0030] As a preferred embodiment of the present invention, the collagen concentration in the surface modifier may be 5 to 100 μg / ml.

[0031] As a preferred embodiment of the present invention, the collagen powder of the surface modifier may include one or more selected from bovine skin-derived collagen, porcine skin-derived collagen, chicken skin-derived collagen, and fish-derived collagen.

[0032] Another objective of the present invention relates to a method for manufacturing an edible support described above, which can be manufactured by performing a process comprising: a first step of preparing a TVP (tissue vegetable protein) raw material; a second step of manufacturing an extruder by performing an extrusion process with the TVP raw material and water; and a third step of forming a collagen modification layer on the surface of the extruder by coating the extruder with a surface modifier, drying at 30 to 40°C, and washing the surface with a PBS (phosphate buffer saline) buffer solution.

[0033] As a preferred embodiment of the present invention, the extruded product of the second stage may have a length of 3 mm or more and a cross-sectional diameter of 9 mm for the long side and 7 mm for the short side before hydration, and a length of 3 mm or more and a cross-sectional diameter of 10 mm for the long side and 8 mm for the short side after hydration. At this time, the hydration is performed for 24 hours under conditions of immersion in water at 20 to 27°C and stirring at 50 rpm using a vertical mixer.

[0034] As a preferred embodiment of the present invention, the surface modifier of step 3 may include collagen powder having a molecular weight of 10 to 250 kDa and acetic acid at a concentration of 0.05 to 1.0 volume%.

[0035] Another objective of the present invention is to provide a method for producing cultured meat by culturing cells on the edible support described above.

[0036] In addition, another objective of the present invention is to provide a processed cultured meat product in which both the cultured meat, in which cells are cultured on the edible support described above, and the edible support are edible. Effects of the invention

[0038] The cultured meat support of the present invention is edible and has a texture similar to meat, so there is no need to separate the cultured cells from the support after the completion of cell culture. Furthermore, since the support of the present invention has a low rate of reduction in compressive strength even after hydration, it not only exhibits excellent shape stability during the cell culture process but also has a very high cell adhesion rate.

[0039] The effects of the present invention are not limited to those mentioned above, and unmentioned effects will be clearly understood by those skilled in the art from the following description. Brief explanation of the drawing

[0041] Figure 1 is a photograph of the edible support prepared in Example 1. Figure 2A is a photograph of the edible support prepared in Comparative Example 1, and B is a photograph of the hydrated edible support. Figure 3 is a photograph of the edible support prepared in Comparative Example 2-1. Figure 4A is a photograph of the edible support prepared in Comparative Example 2-2, and Figure 4B is a photograph of the hydrated edible support. Figure 5A is a photograph of the edible support prepared in Comparative Example 3, and B is a photograph of the hydrated edible support. Figure 6A is a photograph of the edible support prepared in Comparative Example 4, and B is a photograph of the hydrated edible support. Figure 7 is cell proliferation rate measurement data using the edible support of Example 1, Comparative Example 2-1, and Comparative Example 3 carried out in Experimental Example 3. Specific details for implementing the invention

[0042] In the present invention, "textured vegetable protein (TVP) raw material" refers to a raw material (or material) of an extruder used in an extrusion process, and "textured vegetable protein (TVP)" refers to an extruder processed from said raw material.

[0043] The numerical limiting range described in the present invention includes all values ​​within the starting point and ending point ranges, and specifically, for example, the range “20℃ to 100℃” means that it includes not only values ​​of ‘20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, 100℃’ but also any values ​​between integers valid within the described range categories such as 20℃ to 40℃, 50℃ to 70℃, 80℃ to 100℃’. In addition, the range of “10 minutes to 100 minutes” means that it includes, for example, all integers including 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 60 minutes to 70 minutes, 70 minutes to 80 minutes, 80 minutes to 90 minutes, and 90 minutes to 100 minutes, as well as any value between integers valid for the range described as 20 minutes to 40 minutes, 80 minutes to 100 minutes, and so on.

[0044] The present invention will be explained in more detail below through a method for manufacturing an edible support for cultured meat according to the present invention.

[0045] The edible support of the present invention is manufactured by performing a process comprising: a first step of preparing a textured vegetable protein (TVP) raw material; a second step of producing an extruder by performing an extrusion process with water on the textured vegetable protein raw material; and a third step of forming a collagen modification layer on the surface of the extruder by coating the extruder with a surface modifier, followed by drying and surface washing.

[0046] The TVP raw material of step 1 may include one or more selected from isolated soy protein powder, concentrated soy protein powder, isorated soy protein powder, textured wheat protein powder, mung bean protein powder, fava bean protein powder, chickpea protein powder, lupin protein powder, lentil protein powder, wheat gluten powder, sunflower seed isolated protein powder, pumpkin protein powder, rice protein powder, hemp seed protein powder, oat protein powder, potato protein powder, and corn protein powder; preferably, it may include one or more selected from isolated soy protein powder, concentrated soy protein powder, isorated soy protein powder, textured wheat protein powder, fava bean protein powder, and chickpea protein powder; more preferably, it may include one or more selected from isolated soy protein powder, concentrated soy protein powder, and isorated soy protein powder.

[0047] In addition, the above TVP raw material may be used comprising 20.50 to 22.50 wt% carbohydrates, 62.20 to 66.50 wt% crude protein, 1.50 to 2.00 wt% crude fat, and the remaining amount of moisture and ash; preferably, it may be used comprising 21.00 to 22.30 wt% carbohydrates, 62.50 to 66.00 wt% crude protein, 1.60 to 1.95 wt% crude fat, and the remaining amount of moisture and ash; and more preferably, it may be used comprising 21.00 to 22.00 wt% carbohydrates, 62.50 to 65.00 wt% crude protein, 1.60 to 1.90 wt% crude fat, and the remaining amount of moisture and ash, which is advantageous in terms of securing appropriate strength of the edible support that is an extruded product and maintaining strength after hydration.

[0048] Next, Step 2 is a process of manufacturing a scaffold-shaped extruded product by feeding the prepared TVP raw material into an extruder and performing an extrusion process. At this time, no materials other than the TVP raw material are used as raw material fed into the extruder.

[0049] In addition, the extruder mentioned above may be a general extruder used in the industry, for example, a uniaxial extrusion molding machine, a biaxial extrusion molding machine, etc.

[0050] In addition, as a preferred embodiment of the above extrusion process, the process is performed by feeding TVP raw material into the feeding hopper of a twin-screw extrusion machine at a feeding amount of 6.0 to 12.0 kg / hr, preferably 7.0 to 10.0 kg / hr. Furthermore, the process is performed by feeding water into the barrel of the twin-screw extrusion machine at a feeding amount of 3.50 to 5.00 kg / hr, preferably 3.80 to 4.90 kg / hr, and more preferably 4.00 to 4.70 kg / hr, wherein the water fed is water with a temperature of 20 to 25°C. Additionally, the die temperature of the extrusion machine is preferably 140 to 170°C, preferably 145 to 160°C, and more preferably 145 to 155°C.

[0051] At this time, performing the extrusion process such that the ranges of the TVP raw material supply amount, water supply amount, water temperature, and die temperature satisfy the above ranges is advantageous in terms of maintaining appropriate compressive strength after hydration while allowing the extruded product or the edible support manufactured using it to have a meat-like texture and compressive strength. For example, if the water supply amount exceeds 5.00 kg / hr, the porosity of the extruded product becomes too high, which causes a problem of significantly lower compressive strength; if the water supply amount is less than 3.50 kg / hr, the compressive strength is high, but there may be a problem of exceeding the appropriate compression range after hydration. In addition, as the die temperature of the extrusion molding machine increases, the compressive strength of the edible support, which is the extruded product, tends to increase after hydration; however, having the die temperature within the above range is advantageous in terms of securing appropriate compressive strength after hydration.

[0052] In addition, the shape of the extruder can be manufactured in various forms, such as a cylindrical shape or a column with a polygonal cross-section, by adjusting the die hole portion of the extrusion molding machine, and the length and / or cross-sectional diameter of the extruder can also be freely adjusted. The extruder is in the form of a scaffold, and in the present invention, the scaffold refers to an extruder having a porous structure and an internal network structure, having a length of 3 mm or more and a cross-sectional diameter of 9 mm on the long side and 7 mm on the short side before hydration, and a length of 3 mm or more and a cross-sectional diameter of 10 mm on the long side and 8 mm on the short side after hydration. At this time, the hydration is performed for 24 hours under conditions where the extruder is immersed in water at 20~27℃ and stirred at 50 rpm using a vertical mixer.

[0054] Next, step 3 is a process of surface-modifying the surface of the extruder produced in step 2 with collagen.

[0055] The surface modifier of step 3 comprises collagen powder and acetic acid, and can be prepared by dissolving the collagen powder in acetic acid. The surface modifier can be prepared by dissolving the collagen powder in the acetic acid such that the collagen concentration is 5 to 100 μg / ml, preferably 5 to 60 μg / ml, and more preferably 7 to 30 μg / ml. At this time, if the collagen concentration in the surface modifier is less than 5 μg / ml, there may be a problem where the cell adhesion rate is significantly lowered because the amount of collagen fixed on the surface of the surface-treated extruder is small. Even if the collagen concentration exceeds 100 μg / ml, not only is there no further effect of increasing the cell adhesion rate, but rather there may be a problem where cell adhesion is inhibited and the cell proliferation rate is low, making it uneconomical.

[0056] In addition, among the surface modifier components, it is advantageous in terms of cell culture to use collagen powder with a molecular weight of 300 kDa or less, preferably collagen powder with a molecular weight of 10 to 250 kDa.

[0057] In addition, the collagen powder may include one or more types selected from collagen derived from cow skin, collagen derived from pig skin, collagen derived from chicken skin, and collagen derived from fish.

[0058] In addition, among the surface modifier components, the concentration of the acetic acid is preferably 0.05 to 1.0 volume%, preferably 0.05 to 0.5 volume%, and more preferably 0.08 to 0.20 volume%.

[0059] In addition, the third stage of coating treatment is preferably performed through an immersion method in which the extruded product is immersed in a surface modifier, or a spray method in which the surface modifier is sprayed onto the surface of the extruded product.

[0060] Then, in step 3, after the coating treatment as described above, the coating layer is formed by drying it at 30 to 40°C, preferably 35 to 40°C, and then washing is performed with PBS (phosphate buffer saline) buffer solution.

[0062] The edible support for cell culture of the cultured meat of the present invention, manufactured by the above method, is a porous scaffold.

[0063] The edible support of the present invention, hydrated for 24 hours under conditions of immersing in water at 20–27°C and stirring at 50 rpm using a vertical mixer, has a length of 3 mm and a cross-sectional area of ​​251 mm 2 When this occurs, the total average pore area in the hydrated edible support is 10,000 µm. 2 It may be greater than 500,000 µm, preferably 500,000 µm 2 It may be greater than, and more preferably 1,200,000 to 2,300,000 µm 2 It could be.

[0064] In addition, the total average area of ​​pores within the hydrated edible support increases to 10% or less, preferably to 4.0% to 10.0%, and more preferably to 4.0% to 6.5%.

[0065] Also, length 3mm and cross-sectional area 251mm 2 When the compressive strength of the above-mentioned hydrated edible support is measured by compressing it to 75% of its length or height using a Brookfield CTX measuring instrument, the compressive strength may be 100 to 300g.

[0066] In addition, the above edible support has a low reduction rate in compressive strength after hydration, and the compressive strength measured after hydrating the above edible support for 21 days may have a reduction rate in compressive strength according to the following mathematical formula 1 of less than 76.0%, preferably 70 to 76%, and more preferably 70.0 to 75.5%.

[0067] [Mathematical Formula 1]

[0068] Compressive strength reduction rate (%) = (Compressive strength of edible support before hydration - Compressive strength of edible support after 21 days of hydration) / (Compressive strength of edible support before hydration) × 100%

[0069] In mathematical formula 1, the above compressive strength was measured by compressing the edible support to 75% of its length or height using Brookfield’s CTX, and the above hydration was performed for 21 days under conditions where the edible support was immersed in water at 20–27°C and stirred at 50 rpm using a vertical mixer.

[0071] In addition, the edible scaffold of the present invention has a very excellent cell adhesion rate, and by suspension culture of muscle stem cells under conditions of 37°C and 5 volume% CO2, the edible scaffold (length 3 mm and cross-sectional area 251 mm) 2 When measuring the cell adhesion rate for ), the increase rate of the cell adhesion rate can satisfy the cell adhesion rate of Equation 1 below.

[0072] [Equation 1]

[0073] 500% ≤ cell adhesion growth rate ≤ 800%, preferably 550% ≤ cell adhesion growth rate ≤ 780%, more preferably 600% ≤ cell adhesion growth rate ≤ 750%

[0074] In Equation 1, the cell adhesion growth rate is (cell adhesion rate on day 14 / cell adhesion rate on day 7) × 100%.

[0075] In addition, the edible scaffold of the present invention is obtained by suspension culture of muscle stem cells under conditions of 37°C and 5 volume% CO2, thereby producing an edible scaffold (length 3 mm and cross-sectional area 251 mm² 2 When measuring the cell adhesion rate for ), the increase rate of the cell adhesion rate can satisfy the cell adhesion rate of Equation 2 below.

[0076] [Equation 2]

[0077] 600% ≤ cell adhesion growth rate ≤ 1200%, preferably 750% ≤ cell adhesion growth rate ≤ 1200%, more preferably 800% ≤ cell adhesion growth rate ≤ 1150%

[0078] In Equation 2, the cell adhesion growth rate is (cell adhesion rate on day 21 / cell adhesion rate on day 14) × 100%.

[0080] Cultured meat can be produced by culturing various cells using the edible support of the present invention prepared by the method described above, and suitable cells for culture include muscle stem cells, mesenchymal stem cells, adipose stem cells, fibroblasts, embryonic stem cells, induced pluripotent stem cells, vascular stem cells, and / or animal cells.

[0081] In addition, culture conditions and the culture medium used vary depending on the cultured cells, and culture can be performed by selecting generally known culture conditions and a culture medium according to the cultured cells.

[0082] The edible support of the present invention has appropriate strength and can maintain strength that allows for a meat-like texture even after hydration, and can provide various processed cultured meat products in which the edible support and the cultured meat can be consumed together without separating the cultured meat from the support after producing cultured meat by culturing cells on the edible support.

[0084] The present invention is described in detail through preferred embodiments, and the following embodiments are illustrative of the invention and the present application is not limited by the following embodiments.

[0085] [Example]

[0086] Example 1: Preparation of an edible support

[0087] IFF's SPC (ALPHAR ® 12) was prepared as a TVP (TVP) raw material, and the components of SPC (Soy Protein Concentrate) are shown in Table 1 below.

[0088] Next, the TVP raw material is fed into the feeding hopper of the twin-screw extrusion machine, and extrusion is performed under extrusion conditions of a TVP raw material feed rate of 7.50 kg / hr, a water feed rate through the barrel of 4.62 kg / hr, and a die temperature of 158℃ to produce an extruded product, which is then cut to a length of 3 mm and a cross-sectional area of ​​approximately 198 mm 2 TVP, which is a cylindrical extrusion, was manufactured. The number of holes, hole diameter, and hole length (land) design of the die were 2 holes, Φ3mm*2mm.

[0089] Next, the cylindrical extruder TVP was immersed in a surface modifier at 37°C for 60 minutes, then removed, dried at 35–36°C, and surface washed with PBS buffer to produce an edible support having a collagen modified layer formed on the surface of the extruder. At this time, the surface modifier used was prepared by dissolving collagen powder (bovine-derived collagen) with a molecular weight of 200–250 Da in a 0.1 volume% aqueous acetic acid solution to achieve a final concentration of 10 µg / ml.

[0090] And, a photograph of the manufactured edible support is shown in Fig. 1.

[0092] Comparative Example 1

[0093] An edible support was prepared in the same manner as in Example 1 above, but without surface modification, having a length of 3 mm and a cross-sectional area of ​​approximately 198 mm 2 A cylindrical extrusion (size before hydration) was manufactured as a support. A photograph of the same is shown in Fig. 2A.

[0095] Comparative Example 2-1

[0096] Using the commercially available IFF 4380 product, an edible support of the same shape and size was manufactured by surface modification treatment in the same manner as in the example, and this is shown in Fig. 3.

[0098] Comparative Example 2-2

[0099] An edible support was prepared using the same extruder as Comparative Example 2-1, but without surface modification, and the extruder was prepared as an edible support in the same shape and size, and a photograph thereof is shown in FIG. 4A.

[0101] Comparative Example 3

[0102] ADM’s ISP (product name: ArconS) was prepared as a TVP (TVP) raw material, and the composition of the ISP (Isolated Soy Protein) is shown in Table 1 below.

[0103] Next, the TVP raw material is fed into the feeding hopper of the twin-screw extrusion machine, and extrusion is performed under extrusion conditions of a TVP raw material feed rate of 12.00 kg / hr, a water feed rate into the barrel of 5.00 kg / hr, and a die temperature of 170℃ to produce an extruded product, which is then cut to a length of 3 mm and a cross-sectional area of ​​approximately 198 mm 2 A cylindrical extrusion (pre-hydration size) was manufactured.

[0104] Next, the above cylindrical extruded material was immersed in a surface modifier at 37°C for 60 minutes, then removed, dried at 35–36°C, and surface washed with PBS buffer solution to produce an edible support with a modified layer formed on the surface of the extruded material. The same surface modifier as in Example 1 was used.

[0105] A photograph of the manufactured edible support is shown in Fig. 5A.

[0107] Comparative Example 4

[0108] An edible support was manufactured in the same manner as Comparative Example 3 above, but the extrusion molding conditions were performed with a TVP raw material supply amount of 9.00 kg / hr, a water supply amount of 4.54 kg / hr, and a die temperature of 170°C to produce an extruded product of the same size, and then surface modification was performed in the same manner to manufacture an edible support, the photograph of which is shown in FIG. 6A.

[0109] Classification (g / 100g) TVP Raw Material Analysis Example 1, Comparative Example 1 Comparative Example 3, Comparative Example 4 carbohydrate 21.55 19.58 crude protein 63.23 68.02 crude fat 1.72 1.35 Moisture and ash 13.5 11.05

[0111] Experimental Example 1: Compressive Strength Measurement

[0112] The compressive strength of each edible support prepared in Example 1 and Comparative Examples 1 to 4 before and after hydration was measured by the following method, and the results are shown in Table 2 below.

[0113] Hydration was performed by immersing the edible support in water at 20 to 23°C for 30 minutes, 7 days (1 week), 14 days (2 weeks), and 21 days (3 weeks), while stirring with the impeller of a mixer at 50 rpm.

[0114] In addition, a micrograph of the support of Comparative Example 1, which underwent 24 hours of hydration, is shown in FIG. 2B, a micrograph of the support of Comparative Example 2-2 is shown in FIG. 4B, a micrograph of the support of Comparative Example 3 is shown in FIG. 5B, and a micrograph of the support of Comparative Example 4 is shown in FIG. 6B.

[0115] Compressive strength measurement experiments were performed using Brookefield’s CTX equipment. The height (or length) of the sample was measured by moving a ball-type probe vertically, and the sample was compressed to 75% of the measured height (or length), and the value measured at that point was determined as the compressive strength.

[0116] In addition, the reduction rate of compressive strength was calculated based on the following mathematical formula 1.

[0117] [Mathematical Formula 1]

[0118] Compressive strength reduction rate (%) = (Compressive strength of edible support before hydration - Compressive strength of edible support after 21 days of hydration) / (Compressive strength of edible support before hydration) × 100%

[0119] division Example 1 Comparative Example 1 Comparative Example 2-1 Comparative Example 2-2 Comparative Example 3 Comparative Example 4 TVP raw materials IFF's SPC (ALPHAR ® 12) IFF's 4380 ADM's ArconS Presence or absence of surface treatment ○ × ○ × ○ ○ Compressive strength (g) Sign language 809.3 809.3 3867.7 3867.7 1283.4 Unmeasurable 30 minutes of sign language 247.9 274.6 943.3 1172.4 137.18 881.0 Sign language week 1 162.9 242.4 606.5 931.9 138.4 597.3 Sign language 2 weeks 252.5 260.4 705.9 548.3 140.48 601.4 Sign language 3 weeks 202.8 180.8 996.8 579.3 Unmeasurable 490.02 Compression strength reduction rate (%), 3-week hydration 74.71% 77.63% 74.22% 85.80% Uncalculated Uncalculated

[0120] Looking at the compressive strength measurement results in Table 2 above, the reduction rate of compressive strength measured 3 weeks after hydration was less than 75%, showing excellent compressive strength retention. In contrast, Comparative Example 1, which was not surface-treated, showed a tendency for a higher reduction rate of compressive strength compared to Example 1.

[0121] This trend could also be confirmed through the reduction rate of compressive strength in Comparative Example 2-1 and Comparative Example 2-2.

[0122] Also, the appropriate compressive strength for having a meat texture is 100g to 300g after hydration, preferably 200g to 300g, but Comparative Example 2-1 had a problem that the compressive strength measured after 3 weeks of hydration was 900g or more, which was too high.

[0123] In addition, Comparative Example 3 was completely decomposed in the third week of the hydration experiment, and it is determined that it was completely decomposed due to the impact caused by the impeller of the mixer.

[0124] In addition, in the case of Comparative Example 4, there was a problem where the compressive strength before hydration exceeded the measurement limit of 5000g, and this is judged to be the result of inappropriate extrusion process conditions.

[0126] Experimental Example 2: Measurement of the total pore area in an edible support

[0127] The total area of ​​pores within the edible support before and after hydration for each of the edible supports prepared in the above examples and comparative examples was measured by the following method, and the results are shown in Table 3 below.

[0128] The pore area was calculated by manually defining the outlines of all pores present on the plane of the edible support, photographing three supports once with a Nikon SMZ745T using a low-magnification microscope and the compatible program HKBasic, and specifying the area of ​​all pore regions observable in the photograph. However, when selecting pores, visual inspection was performed, and non-pores were excluded from the measurement.

[0129] division Example 1 Comparative Example 2-1 Comparative Example 3 Comparative Example 4 Total pore area within the support before hydration (㎛) 2 ) 2,084,450 Unmeasurable 269,156 113,221 Total pore area (㎛) within the support after 24 hours of hydration 2 ) 2,182,879 362,779 219,725 Growth rate of total pore area 4.72% 34.78% 84.07%

[0130] Looking at Table 3 above, the edible support of the present invention showed no significant change in the total pore area within the support before and after hydration, exhibiting an area change rate of 5% or less.

[0131] In contrast, Comparative Example 3 and Comparative Example 4 showed excessively high area change rates of 34.78% and 84.07%, respectively.

[0132] In addition, Comparative Example 2-1 had a surface formed of micropores, so when photographed with a low-magnification microscope, the pores on the surface could not be distinguished and were photographed as a flat plane, making it impossible to measure the total area of ​​the pores.

[0133] Through this, it was confirmed that the shape stability of the edible support of the present invention is very high.

[0135] Experimental Example 3: Measurement of cell adhesion rate to edible support

[0136] After externally coating the edible supports of Example 1, Comparative Example 2-1, and Comparative Example 3 with collagen, the cell adhesion rate for each of them was measured, and the results are shown in Table 4 below.

[0137] Cell culture was performed using the 3D cell culture method, which involves suspending cells in a medium. The culture medium used was DMEM (Dulbeco's Modified Eagle's Medium) containing FBS (Fetal bovine serum), and the cells used were mouse myoblasts, specifically C2C12. The cell culture was performed at a temperature of 37℃ for 1 week (7 days), 2 weeks (14 days), and 3 weeks (21 days).

[0138] In addition, the cell adhesion rate to the scaffold was measured by detaching and counting cells from the scaffold, and the measurement data is shown in Figure 7, and the unit of the cell adhesion rate in Table 4 is %.

[0139] division Example 1 Comparative Example 2-1 Comparative Example 3 Week 1 Cell Attachment Rate (%) 40.6% 53.0% 2.7% Cell count (Week 1) 20,300 26,500 1,366 Cell count (Week 2) 128,333 79,800 41,566 Cell count (Week 3) 1,442,333 862,333 634,000 Cell proliferation rate (cell increase rate in Week 2 compared to Week 1) 6.3 times 3 times 30 times Cell proliferation rate (cell increase rate in week 3 compared to week 2) 11.2 times 10.8 times 15.3 times

[0140] Looking at the cell adhesion rate measurement results in Table 4 above, Comparative Example 2-1 showed the highest cell adhesion rate but the lowest cell proliferation rate, while Comparative Example 3 showed a low cell adhesion rate but the highest cell proliferation rate.

[0141] Example 1 showed a lower cell adhesion rate than Comparative Example 2-1 but a higher cell proliferation rate, and although the cell proliferation rate was lower than Comparative Example 3, Example 1 showed the highest number of proliferated cells when the total number of cells was counted. This is because the pore size of the support is a factor that ensures the flowability of the medium inside and outside the support and supports the movement of cell waste products; in the case of Comparative Example 2-1 and Comparative Example 3, the micropores resulted in insufficient flowability of the medium and insufficient movement of waste products generated during cell growth, which is believed to have caused the above differences in cell adhesion rate and cell adhesion growth rate.

[0143] Examples 2 to 5 and Comparative Examples 5 to 8

[0144] Examples 2 to 5 and Comparative Examples 5 to 8 were carried out by preparing a cylindrical extruded TVP using the TVP raw material used in Example 1 in the same way, and then treating it with a surface modifier to form a collagen modified layer, thereby producing an edible support of the same size and shape as Example 1, but with different extrusion process conditions as shown in Table 5 below.

[0145] Then, the manufactured edible support (extruder) was immersed in water at 20–23°C and stirred at 50 rpm using a vertical mixer for 24 hours, after which the compressive strength was measured, and the results are shown in Table 5 below.

[0146] division Screw speed (rpm) Odds * Hole Diameter (mm) * Hole Length (mm) Die temperature (°C) Water supply volume (kg / hr) TVP raw material input amount (kg / hr) Compressive strength (g) after 24 hours of hydration Compressive strength (g) Week 1 Week 2 Week 3 Example 2 500 2 holes * 3 * 2 150 4.37 8 277.36 323.12 394.84 159.66 Example 3 500 2 holes * 3 * 2 150 3.98 8 267.74 429.18 395.4 130.84 Example 4 500 2 holes * 3 * 2 160 4.37 8 830.10 874.48 727.12 281.08 Example 5 500 2 holes * 3 * 2 170 4.90 9 280.72 424.22 378.1 198.32 Comparative Example 5 500 2 holes * 3 * 2 150 5.16 8 920.02 1037.98 896.64 385.30 Comparative Example 6 500 2 holes * 3 * 2 170 5.83 9 150.20 229.18 188.62 76.33 Comparative Example 7 500 2 holes * 3 * 2 170 6.52 10 252.32 178.76 254.32 81.04 Comparative Example 8 500 2 holes * 2 * 6 170 5.05 12 145.90 176.28 129.94 50.00

[0147] Looking at Table 5 above, it was confirmed that in the case of Examples 2 to 5, the compressive strength after hydration for 3 weeks satisfied the range of 100 to 300g, which has a meaty texture.

[0148] In contrast, Comparative Example 5, in which the amount of water supplied to the barrel of the twin-screw extrusion molding machine exceeded 5.00 kg / hr, had a problem in which the compressive strength exceeded the 300g range after hydration for 3 weeks, and Comparative Examples 6 and 7, in which the amount of TVP raw material input was slightly increased, the die temperature of the extrusion molding machine was 170℃, and the amount of water supplied exceeded 5.00 kg / hr, had a problem in which the compressive strength actually decreased significantly despite the increase in the amount of water supplied compared to Comparative Example 5.

[0149] In addition, in Comparative Example 8, which had a high amount of TVP raw material input, there was also a problem with significantly low compressive strength after hydration.

[0151] Through the above examples and experimental examples, it was confirmed that the present invention can provide an edible support for cell culture of cultured meat that satisfies the physical properties required for a support used in the production of cultured meat, is edible, has a meat texture, and eliminates the need for separate separation of cultured cells from the support.

Claims

Claim 1 A method for manufacturing an edible support for cell culture of cultured meat, comprising: a first step of preparing a textured vegetable protein (TVP) raw material that does not contain isolated soy protein; and a second step of manufacturing an extruded product by performing an extrusion process on the textured vegetable protein raw material together with water. A method for manufacturing an edible scaffold for cell culture of cultured meat, comprising: a third step of forming a collagen coating layer on the surface of the extruded product to produce an edible scaffold for cell culture of cultured meat; wherein the tissue plant protein raw material comprises 20.50 to 22.50 wt% carbohydrates, 62.20 to 66.50 wt% crude protein, 1.50 to 2.00 wt% crude fat, and the remaining amount of moisture and ash; and wherein the edible scaffold has a compressive strength reduction rate of less than 76.0% according to the following mathematical formula 1: [Mathematical Formula 1] Compressive strength reduction rate (%) = (Compressive strength of edible scaffold before hydration - Compressive strength of edible scaffold after hydration for 21 days) / (Compressive strength of edible scaffold before hydration) × 100% In mathematical formula 1, the compressive strength is measured by compressing the edible scaffold to 75% of its length or height using Brookfield's CTX, and the hydration The process was carried out for 21 days under conditions where an edible support was immersed in water at 20–27°C and stirred at 50 rpm using a vertical mixer. Claim 2 A method for manufacturing an edible support for cell culture of cultured meat, wherein, in claim 1, the extruder produced in the second step has a length of 3 mm or more and a cross-sectional diameter of 9 mm for the long side and 7 mm for the short side before hydration, and after hydration for 24 hours, has a length of 3 mm or more and a cross-sectional diameter of 10 mm for the long side and 8 mm for the short side. Claim 3 A method for manufacturing an edible support for cell culture of cultured meat, wherein, in claim 1, the third step comprises the step of coating the extruded product with a surface modifier comprising collagen powder having a molecular weight of 10 to 250 kDa and acetic acid at a concentration of 0.05 to 1.0 volume%. Claim 4 A method for manufacturing an edible support for cell culture of cultured meat, wherein, in paragraph 3, the third step further comprises the step of forming a collagen modified layer on the surface of the extruder by coating the extruder with the surface modifying agent, drying at 30 to 40°C, and washing the surface with PBS (phosphate buffer saline) buffer solution. Claim 5 A method for preparing an edible support for cell culture of cultured meat, wherein, in paragraph 3, the concentration of the collagen powder in the surface modifier is 5 to 100 μg / ml. Claim 6 A method for manufacturing an edible support for cell culture of cultured meat, wherein, in paragraph 3, the collagen powder comprises one or more types selected from bovine skin-derived collagen, porcine skin-derived collagen, chicken skin-derived collagen, and fish-derived collagen. Claim 7 A method for manufacturing an edible support for cell culture of cultured meat according to claim 1, wherein the total average area of ​​pores within the hydrated edible support, measured after hydrating the edible support manufactured in step 3 for 24 hours, increases to 10% or less compared to the total average area of ​​pores within the edible support before hydration. Claim 8 In claim 7, the hydrated edible support has a length of 3 mm and a cross-sectional area of ​​251 mm 2 When, the total average area of ​​pores in the hydrated edible support is 10,000 μm 2 Lee Sang-in, method for manufacturing an edible support for cell culture of cultured meat. Claim 9 A method for manufacturing an edible support for cell culture of cultured meat, wherein, in claim 7, the compressive strength (TA hardness) is 100 to 300 g when the hydrated edible support is compressed to 75% of its length or height using a Brookfields CTX measuring instrument. Claim 10 A method for manufacturing an edible scaffold for cell culture of cultured meat, wherein, in claim 1, the edible scaffold satisfies the following Equation 1 when measuring the cell adhesion rate of muscle stem cells to the edible scaffold after suspension culture of muscle stem cells at 37°C and 5 vol% CO2: [Equation 1] 500% ≤ (Cell adhesion rate on day 14 / Cell adhesion rate on day 7) × 100% ≤ 800% Claim 11 A method for producing cultured meat by culturing cells on an edible support prepared by the method for producing an edible support for cell culture of cultured meat according to any one of claims 1 to 10. Claim 12 A processed food product of cultured meat, comprising: cultured meat in which cells are cultured on an edible support prepared by the method for preparing an edible support for cell culture of cultured meat according to any one of claims 1 to 10; and said edible support; all of which are edible. Claim 13 delete

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